TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Effect of Ethanol-exposure on Innate Immune Response in Sepsis.

Alcohol use disorder, reported by one in eight critically ill patients, is a risk factor for death in sepsis patients. Sepsis, the leading cause of death kills over 270,000 patients in the United States alone and remains without targeted therapy. Immune response in sepsis transitions from an early hyper-inflammation to persistent inflammation and immunosuppression and multiple organ dysfunction during late sepsis. Innate immunity is the first line of defense against pathogen invasion. Ethanol exposure is known to impair innate and adaptive immune response and bacterial clearance in sepsis patients. Specifically, ethanol-exposure is known to modulate every aspect of innate immune response with and without sepsis. Multiple molecular mechanisms are implicated in causing dysregulated immune response in ethanol-exposure with sepsis, but targeted treatments have remained elusive. In this manuscript, we outline the effects of ethanol-exposure on various innate immune cell types in general and during sepsis.

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction and senescence by inhibiting sirtuin-mediated deacetylation.

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD+ /NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD+ -dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD+ -dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD+ , that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD+ -sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD+ content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD+ biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues.

Hemoglobin resident in the lung epithelium is protective for smooth muscle soluble guanylate cyclase function.

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1ß1 heterodimerization. Silencing Hbαß in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived.

Hepatic kinome atlas: An in-depth identification of kinase pathways in liver fibrosis of humans and rodents.

BACKGROUND AND AIMS: Resolution of pathways that converge to induce deleterious effects in hepatic diseases, such as in the later stages, have potential antifibrotic effects that may improve outcomes. We aimed to explore whether humans and rodents display similar fibrotic signaling networks. APPROACH AND RESULTS: We assiduously mapped kinase pathways using 340 substrate targets, upstream bioinformatic analysis of kinase pathways, and over 2000 random sampling iterations using the PamGene PamStation kinome microarray chip technology. Using this technology, we characterized a large number of kinases with altered activity in liver fibrosis of both species. Gene expression and immunostaining analyses validated many of these kinases as bona fide signaling events. Surprisingly, the insulin receptor emerged as a considerable protein tyrosine kinase that is hyperactive in fibrotic liver disease in humans and rodents. Discoidin domain receptor tyrosine kinase, activated by collagen that increases during fibrosis, was another hyperactive protein tyrosine kinase in humans and rodents with fibrosis. The serine/threonine kinases found to be the most active in fibrosis were dystrophy type 1 protein kinase and members of the protein kinase family of kinases. We compared the fibrotic events over four models: humans with cirrhosis and three murine models with differing levels of fibrosis, including two models of fatty liver disease with emerging fibrosis. The data demonstrate a high concordance between human and rodent hepatic kinome signaling that focalizes, as shown by our network analysis of detrimental pathways. CONCLUSIONS: Our findings establish a comprehensive kinase atlas for liver fibrosis, which identifies analogous signaling events conserved among humans and rodents.

SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.

Sirtuin 2 Dysregulates Autophagy in High-Fat-Exposed Immune-Tolerant Macrophages.

Obesity increases morbidity and resource utilization in sepsis patients. The immune response in sepsis transitions from an endotoxin-responsive hyper- to an endotoxin-tolerant hypo-inflammatory phase. The majority of sepsis mortality occurs during hypo-inflammation. We reported prolonged hypo-inflammation with increased sirtuin 2 (SIRT2) expression in obese-septic mice. The effect of direct exposure to high-fat/free fatty acid (FFA) and the role of SIRT2 in immune cells during the transition to hypo-inflammation is not well-understood. Autophagy, a degradation process of damaged protein/organelles, is dysregulated during sepsis. Here, we investigated the effect of direct FFA exposure and the role of SIRT2 expression on autophagy as macrophages transition from hyper-to hypo-inflammation. We found, FFA-exposed RAW 264.7 cells with lipopolysaccharide (LPS) stimulation undergo endotoxin-sensitive ("sensitive") hyper- followed by endotoxin tolerant ("tolerant") hypo-inflammatory phases; SIRT2 expression increases significantly in tolerant cells. Autophagy proteins LC3b-II, and beclin-1 increase in FFA-sensitive and decrease in tolerant cells; p62 expressions continue to accumulate in tolerant cells. We observed that SIRT2 directly deacetylates α-tubulin and impairs autophagy clearance. Importantly, we find SIRT2 inhibitor AK-7 treatment during endotoxin tolerant phase reverses autophagy dysregulation with improved autophagy clearance in FFA-tolerant cells. Thus, we report impaired autophagosome formation and autophagy clearance via increased SIRT2 expression in FFA-exposed tolerant macrophages.

Differential role of MLKL in alcohol-associated and non-alcohol-associated fatty liver diseases in mice and humans.

Hepatocellular death contributes to progression of alcohol-associated (ALD-associated) and non-alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain-like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl-/- mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.

Ethanol Exposure Attenuates Immune Response in Sepsis via Sirtuin 2 Expression.

BACKGROUND: Sepsis and septic shock kill over 270,000 patients per year in the United States. Sepsis transitions from a hyper-inflammatory to a hypo-inflammatory phase. Alcohol dependence is a risk factor for mortality from sepsis. Ethanol (EtOH) exposure impairs pathogen clearance through mechanisms that are not fully understood. Sirtuin 2 (SIRT2) interferes with pathogen clearance in immune cells but its role in the effects of EtOH on sepsis is unknown. We studied the effect of EtOH exposure on hyper- and hypo-inflammation and the role of SIRT2 in mice. METHODS: We exposed C57Bl/6 (WT) mice to EtOH via drinking water and used intraperitoneal cecal slurry (CS)-induced sepsis to study: (i) 7-day survival, (ii) leukocyte adhesion (LA) in the mesenteric microcirculation during hyper- and hypo-inflammation, (iii) peritoneal cavity bacterial clearance, and (iv) SIRT2 expression in peritoneal macrophages. Using EtOH-exposed and lipopolysaccharide (LPS)-stimulated RAW 264.7 (RAW) cell macrophages for 4 hours or 24 hours, we studied: (i) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and SIRT2 expression, and (ii) the effect of the SIRT2 inhibitor AK-7 on inflammatory response at 24 hours. Lastly, we studied the effect of EtOH on sepsis in whole body Sirt2 knockout (SIRT2KO) mice during hyper- and hypo-inflammation, bacterial clearance, and 7-day survival. RESULTS: WT EtOH-sepsis mice showed: (i) Decreased survival, (ii) Muted LA in the microcirculation, (iii) Lower plasma TNF-α and IL-6 expression, (iv) Decreased bacterial clearance, and (v) Increased SIRT2 expression in peritoneal macrophages versus vehicle-sepsis. EtOH-exposed LPS-stimulated RAW cells showed: (i) Muted TNF-α, IL-6, and increased IL-10 expression at 4 hours, (ii) endotoxin tolerance at 24 hours, and (iii) reversal of endotoxin tolerance with the SIRT2 inhibitor AK-7. EtOH-exposed SIRT2KO-sepsis mice showed greater 7-day survival, LA, and bacterial clearance than WT EtOH-sepsis mice. CONCLUSION: EtOH exposure decreases survival and reduces the inflammatory response to sepsis via increased SIRT2 expression. SIRT2 is a potential therapeutic target in EtOH with sepsis.

Sirtuins and Sepsis: Cross Talk between Redox and Epigenetic Pathways.

Sepsis and septic shock are the leading causes of death among hospitalized patients in the US. The immune response in sepsis transitions from a pro-inflammatory and pro-oxidant hyper-inflammation to an anti-inflammatory and cytoprotective hypo-inflammatory phase. While 1/3rd sepsis-related deaths occur during hyper-, a vast majority of sepsis-mortality occurs during the hypo-inflammation. Hyper-inflammation is cytotoxic for the immune cells and cannot be sustained. As a compensatory mechanism, the immune cells transition from cytotoxic hyper-inflammation to a cytoprotective hypo-inflammation with anti-inflammatory/immunosuppressive phase. However, the hypo-inflammation is associated with an inability to clear invading pathogens, leaving the host susceptible to secondary infections. Thus, the maladaptive immune response leads to a marked departure from homeostasis during sepsis-phases. The transition from hyper- to hypo-inflammation occurs via epigenetic programming. Sirtuins, a highly conserved family of histone deacetylators and guardians of homeostasis, are integral to the epigenetic programming in sepsis. Through their anti-inflammatory and anti-oxidant properties, the sirtuins modulate the immune response in sepsis. We review the role of sirtuins in orchestrating the interplay between the oxidative stress and epigenetic programming during sepsis.

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis.

BACKGROUND & AIMS: Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. METHODS: Rip3+/+, Rip3-/-, Mlkl+/+ and Mlkl-/- mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). RESULTS: The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl-/- mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. CONCLUSIONS: Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. LAY SUMMARY: Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

Dietary Synbiotic Supplementation Protects Barrier Integrity of Hepatocytes and Liver Sinusoidal Endothelium in a Mouse Model of Chronic-Binge Ethanol Exposure.

Alcohol overconsumption disrupts the gut microbiota and intestinal barrier, which decreases the production of beneficial microbial metabolic byproducts and allows for translocation of pathogenic bacterial-derived byproducts into the portal-hepatic circulation. As ethanol is known to damage liver sinusoidal endothelial cells (LSEC), here we evaluated dietary supplementation with a previously studied synbiotic on gut microbial composition, and hepatocyte and LSEC integrity in mice exposed to ethanol. We tested a chronic-binge ethanol feeding mouse model in which C57BL/6 female mice were fed ethanol (5% vol/vol) for 10 days and provided a single ethanol gavage (5 g/kg body weight) on day 11, 6 h before euthanasia. An ethanol-treatment group also received oral supplementation daily with a synbiotic; and an ethanol-control group received saline. Control mice were pair-fed and isocalorically substituted maltose dextran for ethanol over the entire exposure period; they received a saline gavage daily. Ethanol exposure decreased gut microbial abundance and diversity. This was linked with diminished expression of adherens junction proteins in hepatocytes and dysregulated expression of receptors for advanced glycation end-products; and this coincided with reduced expression of endothelial barrier proteins. Synbiotic supplementation mitigated these effects. These results demonstrate synbiotic supplementation, as a means to modulate ethanol-induced gut dysbiosis, is effective in attenuating injury to hepatocyte and liver endothelial barrier integrity, highlighting a link between the gut microbiome and early stages of acute liver injury in ethanol-exposed mice.

Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High-Fat Diet-Induced Liver Injury.

Interferon regulatory factor 3 (IRF3) has both transcriptional and nontranscriptional functions. Transcriptional activity is dependent on serine phosphorylation of IRF3, while transcription-independent IRF3-mediated apoptosis requires ubiquitination. IRF3 also binds to inhibitor of nuclear factor kappa B kinase (IKKß) in the cytosol, restricting nuclear translocation of p65. IRF3-deficient mice are highly sensitive to high-fat diet (HFD)-induced liver injury; however, it is not known if transcriptional and/or nontranscriptional activity of IRF3 confers protection. Using a mouse model only expressing nontranscriptional functions of IRF3 (Irf3 S1/S1), we tested the hypothesis that nontranscriptional activity of IRF3 protects mice from HFD-induced liver injury. C57BL/6, Irf3 -/-, and Irf3 S1/S1 mice were fed an HFD for 12 weeks. In C57BL/6 mice, the HFD increased expression of interferon (IFN)-dependent genes, despite a decrease in IRF3 protein in the liver. The HFD had no impact on IFN-dependent gene expression Irf3 -/- or Irf3 S1/S1 mice, both lacking IRF3 transcriptional activity. Liver injury, apoptosis, and fibrosis were exacerbated in Irf3 -/- compared to C57BL/6 mice following the HFD; this increase was ameliorated in Irf3 S1/S1 mice. Similarly, expression of inflammatory cytokines as well as numbers of neutrophils and infiltrating monocytes was increased in Irf3 -/- mice compared to C57BL/6 and Irf3 S1/S1 mice. While the HFD increased the ubiquitination of IRF3, a response associated with IRF3-mediated apoptosis, in Irf3 S1/S1 mice, protection from liver injury was not due to differences in apoptosis of hepatocytes or immune cells. Instead, protection from HFD-induced liver injury in Irf3 S1/S1 mice was primarily associated with retardation of nuclear translocation of p65 and decreased expression of nuclear factor kappa B (NFκB)-dependent inflammatory cytokines. Conclusion: Taken together, these data identify important contributions of the nontranscriptional function of IRF3, likely by reducing NFκB signaling, in dampening the hepatic inflammatory environment in response to an HFD.

A Designer Synbiotic Attenuates Chronic-Binge Ethanol-Induced Gut-Liver Injury in Mice.

Gut dysbiosis and altered short-chain fatty acids are associated with ethanol-induced liver injury. SCFA are fermentation byproducts of the gut microbiota known to have many beneficial biological effects. We tested if a designer synbiotic could protect against ethanol-induced gut-liver injury. C57BL/6 female mice were exposed to chronic-binge ethanol feeding consisting of ethanol (5% vol/vol) for 10 days, followed by a single gavage (5 g/kg body weight) 6 h before euthanasia. A group of mice also received oral supplementation daily with a designer synbiotic, and another group received fecal slurry (FS); control animals received saline. Control mice were isocalorically substituted maltose dextran for ethanol over the entire exposure period. Ethanol exposure reduced expression of tight junction proteins in the proximal colon and induced hepatocyte injury and steatosis. Synbiotic supplementation not only mitigated losses in tight junction protein expression, but also prevented ethanol-induced steatosis and hepatocyte injury. Ethanol exposure also increased hepatic inflammation and oxidative stress, which was also attenuated by synbiotic supplementation. Mice receiving FS were not protected from ethanol-induced liver injury or steatosis. Results were associated with luminal SCFA levels and SCFA transporter expression in the proximal colon and liver. These results indicate supplementation with a designer synbiotic is effective in attenuating chronic-binge ethanol-induced gut-liver injury and steatosis in mice, and highlight the beneficial effects of the gut microbial fermentation byproducts.

The Role of the Gut Microbiome in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, with prevalence increasing in parallel with the rising incidence in obesity. Believed to be a "multiple-hit" disease, several factors contribute to NAFLD initiation and progression. Of these, the gut microbiome is gaining interest as a significant factor in NAFLD prevalence. In this paper, we provide an in-depth review of the progression of NAFLD, discussing the mechanistic modes of hepatocyte injury and the potential role for manipulation of the gut microbiome as a therapeutic strategy in the prevention and treatment of NAFLD.

Complement Factor D protects mice from ethanol-induced inflammation and liver injury.

Complement plays a crucial role in microbial defense and clearance of apoptotic cells. Emerging evidence suggests complement is an important contributor to alcoholic liver disease. While complement component 1, Q subcomponent (C1q)-dependent complement activation contributes to ethanol-induced liver injury, the role of the alternative pathway in ethanol-induced injury is unknown. Activation of complement via the classical and alternative pathways was detected in alcoholic hepatitis patients. Female C57BL/6J [wild type (WT)], C1q-deficient ( C1qa-/-, lacking classical pathway activation), complement protein 4-deficient ( C4-/-, lacking classical and lectin pathway activation), complement factor D-deficient ( FD-/-, lacking alternative pathway activation), and C1qa/FD-/- (lacking classical and alternative pathway activation) mice were fed an ethanol-containing liquid diet or pair-fed control diet for 4 or 25 days. Following chronic ethanol exposure, liver injury, steatosis, and proinflammatory cytokine expression were increased in WT but not C1qa-/-, C4-/-, or C1qa/FD-/- mice. In contrast, liver injury, steatosis, and proinflammatory mediators were robustly increased in ethanol-fed FD-/- mice compared with WT mice. Complement activation, assessed by hepatic accumulation of C1q and complement protein 3 (C3) cleavage products (C3b/iC3b/C3c), was evident in livers of WT mice in response to both short-term and chronic ethanol. While C1q accumulated in ethanol-fed FD-/- mice (short term and chronic), C3 cleavage products were detected after short-term but not chronic ethanol. Consistent with impaired complement activation, chronic ethanol induced the accumulation of apoptotic cells and fibrogenic responses in the liver of FD-/- mice. These data highlight the protective role of complement factor D (FD) and suggest that FD-dependent amplification of complement is an adaptive response that promotes hepatic healing and recovery in response to chronic ethanol. NEW & NOTEWORTHY Complement, a component of the innate immune system, is an important pathophysiological contributor to ethanol-induced liver injury. We have identified a novel role for factor D, a component of the alternative pathway, in protecting the liver from ethanol-induced inflammation, accumulation of apoptotic hepatocytes, and profibrotic responses. These data indicate a dual role of complement with regard to inflammatory and protective responses and suggest that accumulation of apoptotic cells impairs hepatic healing/recovery during alcoholic liver disease.

Faecalibacterium prausnitzii and a Prebiotic Protect Intestinal Health in a Mouse Model of Antibiotic and Clostridium difficile Exposure.

BACKGROUND: Clostridium difficile (CD) infection (CDI) increases patient morbidity, mortality and healthcare costs. Antibiotic treatment induces gut dysbiosis and is both a major risk factor for CD colonization and treatment of CDI. Probiotics have been trialed to support commensal gut microbiota and reduce CDI. This study investigated commensal microbe Faecalibacterium prausnitzii (FP) and a prebiotic, both known to yield butyrate and be anti-inflammatory and immunomodulatory, on CD colonization and gut integrity in mice. METHODS: Mice were randomly grouped and supplemented daily with FP, prebiotic, FP + prebiotic, FP/prebiotic supernatant, or saline throughout the entire study. Following treatment with clindamycin for 3 days, mice were exposed to CD. Feces were collected at baseline, the day after antibiotic, and 1, 3, and 5 days after CD exposure and cultured for bacterial overgrowth and CD colonization. On days 1 and 5 after CD exposure, mice were randomly euthanized, and proximal colon was dissected for histological analysis and preparation of RNA for analysis of proinflammatory and anti-inflammatory cytokines. RESULTS: Although all mice exhibited bacterial overgrowth and CD colonization, bacterial burden resolved quicker in the FP + prebiotic group. This was associated with induction and resolution of innate immune responses, anion exchanger, and tight junction protein preservation in proximal colon. CD toxin virulence potential was questionable as expression of CD toxin B receptor was depleted in the FP + prebiotic group. CONCLUSION: Supplementation with anti-inflammatory butyrate-supporting commensal bacteria and prebiotic may support innate immune responses and minimize bacterial burden and negative effects during antibiotic and CD exposure.

Hyaluronic acid 35 normalizes TLR4 signaling in Kupffer cells from ethanol-fed rats via regulation of microRNA291b and its target Tollip.

TLR4 signaling in hepatic macrophages is increased after chronic ethanol feeding. Treatment of hepatic macrophages after chronic ethanol feeding with small-specific sized hyaluronic acid 35 (HA35) normalizes TLR4 signaling; however, the mechanisms for HA35 action are not completely understood. Here we used Next Generation Sequencing of microRNAs to identify negative regulators of TLR4 signaling reciprocally modulated by ethanol and HA35 in hepatic macrophages. Eleven microRNAs were up-regulated by ethanol; only 4 microRNAs, including miR291b, were decreased by HA35. Bioinformatics analysis identified Tollip, a negative regulator of TLR4, as a target of miR291b. Tollip expression was decreased in hepatic macrophages from ethanol-fed rats, but treatment with HA35 or transfection with a miR291b hairpin inhibitor restored Tollip expression and normalized TLR4-stimulated TNFα expression. In peripheral blood monocytes isolated from patients with alcoholic hepatitis, expression of TNFα mRNA was robustly increased in response to challenge with lipopolysaccharide. Importantly, pre-treatment with HA35 reduced TNFα expression by more than 50%. Taken together, we have identified miR291b as a critical miRNA up-regulated by ethanol. Normalization of the miR291b → Tollip pathway by HA35 ameliorated ethanol-induced sensitization of TLR4 signaling in macrophages/monocytes, suggesting that HA35 may be a novel therapeutic agent in the treatment of ALD.